Live cell imaging with chemical specificity using dual frequency CARS microscopy

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Live cell imaging traditionally relies on fluorescent probes, which can be invasive and affect cellular function.
• Studying lipid dynamics in live cells is challenging due to difficulties in producing effective fluorescent markers and potential labeling artifacts.
• There is a significant need for noninvasive techniques to study cellular and tissue dynamics without chemical or genetic probes.

Purpose of the Study:

• To describe a cost-effective and alignment-insensitive experimental setup for Coherent Anti-Stokes Raman Scattering (CARS) microscopy of lipid droplets in live cells.
• To demonstrate the feasibility of using femtosecond lasers for CARS microscopy, enabling integration with other multiphoton techniques.
• To present a dual-frequency CARS system for enhanced sensitivity and contrast by eliminating background signals.

Main Methods:

• Development of a CARS microscopy setup utilizing spectrally focused femtosecond lasers for vibrational excitation.
• Implementation of a dual-frequency CARS system to suppress nonresonant background signals.
• Application of the developed CARS system for imaging lipid droplets in live cells.

Main Results:

• Femtosecond lasers were effectively spectrally focused for CARS, achieving vibrational excitation comparable to picosecond lasers in a cost-effective manner.
• The dual-frequency CARS system successfully eliminated nonresonant background, leading to superior sensitivity and image contrast.
• The setup allows for label-free, noninvasive imaging and quantification of lipid droplets in live cells.

Conclusions:

• The described CARS microscopy setup, using cost-effective femtosecond lasers, provides a powerful tool for label-free lipid imaging in live cells.
• Integration of CARS with other multiphoton techniques like two-photon fluorescence is facilitated by this approach, paving the way for multimodal instruments.
• This technique overcomes limitations of traditional fluorescence microscopy for lipid studies, offering enhanced sensitivity and accuracy in live-cell dynamics.